Acridinium esters (AE) have provided for an extremely sensitive method of detection and have been used extensively as chemiluminescent labels in both immuno- and nucleic acid assays. Hydrolytically stable, Polysubstituted Aryl Acridinium Esters (PAAE) have proven useful as analytical labels (U.S. Pat. Nos. 4,745,181; 4,918,192; and 5,110,932) with a variety of linkages (U.S. Pat. Nos. 5,241,070; 5,538,901; and 5,663,074) and were the first chemiluminescent, acridinium compounds to satisfy the stringent requirements of commercial ligand binding assays. The utility of PAAE was further enhanced with the advent of Functionalized Hydrophilic PAAE (U.S. Pat. No. 5,656,426) which increased the quantum yield of PAAE and enhanced the performance of PAAE-labeled binding partners in terms of the observed signal to noise ratios and the sensitivities of various binding assays. Additionally, introduction of ionizable groups at the phenoxy moiety produced another sub-class of hydrophilic PAAE (U.S. Pat. Nos. 5,227,489; 5,449,556; and 5,595,875).
M. Kawaguichi, et al. (Bioluminescence and Chemiluminescence, Proceedings of 9th International Symposium 1996, Ed. Hastings, Kricka and Stanley, John Wiley xcexcSons, 1997, pp. 480-484) have described stabilized phenyl acridinium esters for chemiluminescent immunoassays. AE derivatives with additional methyl substitutions at C-1, which are optional at C-3 of the acridinium nucleus with matching mono- or di-methyl substitutions at the ortho-positions of the phenoxy moiety, were shown to have excellent stability in aqueous solution.
EP 0324,202 A1 and subsequently EP 0609,885 A1 both describe acridinium esters with functional groups substituted at the nitrogen atom of the acridinium nucleus. The latter application further describes alternate substituents such as the biphenyl or naphthyl moieties as possible replacements for the phenyl group.
Mattingly, et al. (U.S. Pat. Nos. 5,468,646 and 5,543,524) describe chemiluminescent acridinium salts, and their applications in immunoassays. These acridinium salts belong to another class of compounds termed acridinium sulfonylamides (or N-sulfonylacridinium carboxamides). The acridinium sulfonylamides (AS) have aqueous stabilities which are comparable with PAAE. Mattingly, et al. further describe and claim the analogous chemiluminescent phenanthridinium salts, and their applications in immunoassays, in U.S. Pat. Nos. 5,545,739; 5,565,570, and 5,669,819. Additionally, in these patents a general structure of acridinium sulfonylamides is described showing possible substitutents of a Markush group at the acridinium nucleus.
Conventional acridinium compounds, such as those described in the aforementioned patents and literature, emit light with maxima at about 428 nm upon reaction with hydrogen peroxide in strong alkaline solution. Acridinium compounds which emit light of wavelength maxima  greater than 500 nm have also been described in the prior arts. U.S. Pat. Nos. 5,395,752; 5,702,887 and 5,879,894 describe novel, long-emission acridinium esters (LEAE), where a fused, benzacridinium system is employed to extend the wavelength of emission of the acridinium ester. In the copending PCT application PCT/IB98/00831 Jiang et al. have further extended the PAAE emission maxima well into the region of 600-700 nm by utilizing the principle of energy transfer. This entailed the covalent coupling of luminophores to acridinium ester. When the chemiluminescent reactions of these conjugates were initiated by treatment with alkaline peroxide, light emission was observed at long wavelengths where the wavelength maxima depended upon the structure of the luminophore. In the more recent copending PCT application PCT/US99/18076, Natrajan et al. describe novel acridinium compounds that have intrinsic emission maxima close to or in the near infrared region ( greater than 590 nm). The structural requirements for such long wavelength-emitting acridinium compounds are disclosed.
N-Alkylacridan esters obtained from the reduction of acridinium esters have been used as enzyme substrate indicators for the determination of phosphatases and oxidases and their substrates or products. N-alkylacridan phosphate esters have been engineered as substrates for the direct detection of minute concentrations of alkaline phosphatase (Akhavan-Tafti, H et al. xe2x80x9cLumagen(trademark) APS: New Substrates for the Chemiluminescent Detection of Phosphatase Enzymesxe2x80x9d; Proc. 9th. Internat""l. Symp. Bioluminescence and Chemiluminescence; (1996); Hastings, J. W.; Kricka, L. J.; Stanley; P. E., (Eds.); John Wiley and Sons, Inc., New York, N.Y.; pp. 311-314). Similarly, N-alkylacridancarboxylate esters have been applied as oxidizable indicators for horseradish peroxidase, where the chemiluminescence from the oxidized acridinium ester product was used to quantify either horseradish peroxidase or oxidases and their substrates in coupled enzymatic reactions (Akhavan-Tafti, H et al.; Chemiluminescent Detection of Oxidase Enzymes by Peroxidase-mediated Oxidation of Acridan Compounds; Proc. 9th. Internat""l. Symp. Bioluminescence and Chemiluminescence; (1996); Hastings, J. W.; Kricka, L. J.; Stanley; P. E., (Eds.); John Wiley xcexcSons, Inc., New York, N.Y.; pp. 501-504).
Luminols along with peroxidase have been used as chemiluminescent detectors of hydrogen peroxide generated from dehydrogenase and its cofactors (WO 95/29255).
Various classes of chromogenic, hydride-reducible indicators have been described (Ottaway, J. M.; Oxidation-Reduction Indicators; Internat""l Ser. Monographs Anal. Chem.; (1968); Belcher, R.; Frieser, H., (Eds.); Plenum; pp. 469-529) (Bird, C. L.; Kuhn, A. T.; Electrochemistry of the Viologens; Chem. Soc. Rev.; (1981), 10, pp. 49-82). Several of these, including the oxidized salts of phenazines, phenoxazines and phenothiazines, have been used as chromogenic indicators of hydride from the reduced nicotinamide cofactors (dihydronicotinamide adenine dinucleotide, NADH, and dihydronicotinamide adenine dinucleotide phosphate, NADPH) and the reduced flavin cofactors (dihydroflavin mononucleotide, FMNH2 and dihydroflavin adenine dinucleotide, FADH2) generated by the enzymatic activity of dehydrogenases (Czerlinski, G. H., et al., xe2x80x9cCoupling of Redox Indicator Dyes into an Enzymatic Reaction Cyclexe2x80x9d, J. Biochem. Biophys. Methods, (1988) 15, pp. 241-248) (Nakamura, S., et al., xe2x80x9cUse of 1-Methoxy-5-methylphenaziniummethylsulfate in the Assay of Some Enzymes of Diagnostic Importancexe2x80x9d, Clin. Chim. Acta, (1980), 101, p. 321).
The present invention discloses a method for the measurement of hydride using a chemiluminescent compound. The preferred chemiluminescent molecule is an acridinium compound. The source of hydride for the reduction of acridinium compound may be of chemical or biochemical origin, or the result of enzymatic catalysis. The chemical source of hydride, for example, might be metal hydrides, such as NaBH4. A biochemical source of hydride might be that derived from NADH, NADPH, FMNH2 or FADH2, while an enzymatic source would be the class of oxidoreductases termed dehydrogenases which convert in redox reactions NADH, NADPH, FMNH2 or FADH2 from NAD, NADP, FMN or FAD.
There are numerous potential applications for acridinium compounds as chemiluminescent indicators of hydride. Any applied tests or diagnostic assays, in which hydride is either present at the onset of or generated through the course of a reaction, would benefit from the present invention. Such tests, which could encompass many different formats, as discussed below in detail, may involve the quantitation or detection of metal hydrides, or enzyme cofactors such as NADH, NADPH, FMNH2, or FADH2. Of particular importance, are those diagnostic assays which might use dehydrogenases as reagents, indicators, diagnostic markers or as labels. Ethanol, for example, might be detected with acridinium ester chemiluminescence through the reaction of alcohol dehydrogenase on ethanol, said reaction producing NADH. As a label, dehydrogenase might be used in an ELISA for the detection of a specific analyte with acridinium ester providing the signaling response. Nucleic acid assays using dehydrogenase as a label are also envisioned. Assays for the detection of clinically relevant dehydrogenases such as elevated glutamate dehydrogenase as an indicator of hepatocellular damage might also be developed.